Herbert Zimmermann - Academia.edu (original) (raw)
Papers by Herbert Zimmermann
Purinergic Signalling
Geoffrey Burnstock will be remembered as the scientist who set up an entirely new field of interc... more Geoffrey Burnstock will be remembered as the scientist who set up an entirely new field of intercellular communication, signaling via nucleotides. The signaling cascades involved in purinergic signaling include intracellular storage of nucleotides, nucleotide release, extracellular hydrolysis, and the effect of the released compounds or their hydrolysis products on target tissues via specific receptor systems. In this context ectonucleotidases play several roles. They inactivate released and physiologically active nucleotides, produce physiologically active hydrolysis products, and facilitate nucleoside recycling. This review briefly highlights the development of our knowledge of two types of enzymes involved in extracellular nucleotide hydrolysis and thus purinergic signaling, the ectonucleoside triphosphate diphosphohydrolases, and ecto-5′-nucleotidase.
Purinergic Signalling
Professor Geoffrey Burnstock, a great luminary in science and the founder of our field of puriner... more Professor Geoffrey Burnstock, a great luminary in science and the founder of our field of purinergic signalling, sadly but peacefully died in Melbourne, Australia, on the 2nd of June, 2020, at the age of 91. He had retired at the age of 88, in October 2017, when, after 42 years in London, he decided to fulfill his wife's and family's desire to move back to Australia, where, in 1959, he had been appointed Senior Lecturer at Melbourne University, his first important academic engagement. Geoffrey Burnstock grew up in London where he was born on May 10, 1929. After completing his secondary education at Greenford County Grammar School in 1946, he did his National service with the Air Force in 1947. In 1953, he completed a BSc degree at King's College, University of London, majoring in mathematics and physics. He subsequently completed a PhD at King's College and University College London in the field of zoology, studying gut motility in fish (1957). This turned out to be a great start for the young scientist as his first paper ever was published in Nature. In 1956, he moved to the Physiology Department at the National Institute for Medical Research in Mill Hill, London, as a postdoctoral fellow with Wilhelm Feldberg (1956-1957). There, he developed a novel technique for recording membrane activities from smooth muscle, the "sucrose gap technique." This led to a position with Edith Bulbring in the
Frontiers in Cellular Neuroscience
In mammalian species, including humans, the hippocampal dentate gyrus (DG) is a primary region of... more In mammalian species, including humans, the hippocampal dentate gyrus (DG) is a primary region of adult neurogenesis. Aberrant adult hippocampal neurogenesis is associated with neurological pathologies. Understanding the cellular mechanisms controlling adult hippocampal neurogenesis is expected to open new therapeutic strategies for mental disorders. Microglia is intimately associated with neural progenitor cells in the hippocampal DG and has been implicated, under varying experimental conditions, in the control of the proliferation, differentiation and survival of neural precursor cells. But the underlying mechanisms remain poorly defined. Using fluorescent in situ hybridization we show that microglia in brain express the ADP-activated P2Y 13 receptor under basal conditions and that P2ry13 mRNA is absent from neurons, astrocytes, and neural progenitor cells. Disrupting P2ry13 decreases structural complexity of microglia in the hippocampal subgranular zone (SGZ). But it increases progenitor cell proliferation and new neuron formation. Our data suggest that P2Y 13 receptor-activated microglia constitutively attenuate hippocampal neurogenesis. This identifies a signaling pathway whereby microglia, via a nucleotide-mediated mechanism, contribute to the homeostatic control of adult hippocampal neurogenesis. Selective P2Y 13 R antagonists could boost neurogenesis in pathological conditions associated with impaired hippocampal neurogenesis.
e-Neuroforum
ZusammenfassungATP ist eines der vielseitigsten zellulären Moleküle überhaupt. Seine Rolle als ex... more ZusammenfassungATP ist eines der vielseitigsten zellulären Moleküle überhaupt. Seine Rolle als extrazellulärer Signalstoff wurde erst in den 90er Jahren des letzten Jahrhunderts konsolidiert, mit der Klonierung der Rezeptoren (P2-Rezeptoren) und von Enzymen (Ekto- Nukleotidasen), die extrazelluläres ATP hydrolysieren können. P2-Rezeptoren können je nach Subtyp durch ATP oder durch ADP, UTP, UDP und verschiedene Diadenosinpolyphosphate aktiviert werden. ATP und andere Nukleotide werden in Subtypen synaptischer Vesikel gespeichert und können exozytotisch freigesetzt werden. Außerhalb der Zelle werden Nukleotide bis zum jeweiligen Nukleosid hydrolysiert. Extrazellulär gebildetes Adenosin aktiviert seinerseits P1-Rezeptoren und wird anschließend wieder in die Nervenendigung oder in benachbarte Zellen aufgenommen. Bis jetzt wurden mehrere Ekto-Nukleotidasen kloniert und funktionell charakterisiert. Gegenwärtig analysieren wir ihre unterschiedliche zelluläre Lokalisierung im zentralen und...
Developmental Brain Research, Aug 28, 1995
The distribution of 5'-nucleotidase was investigated in the developing mouse retina and a... more The distribution of 5'-nucleotidase was investigated in the developing mouse retina and also in the retina of the adult mouse and rat using a monospecific antibody directed against 5'-nucleotidase isolated from bovine brain. Already at the stage of the formation of the eye cup strong immunofluorescence was found at the ventricular surface. Throughout development the enzyme remained associated with the outer surface of the retina that corresponds to the former inner surface of the eye vesicle. During embryonic development immunoreactivity was also associated with the proliferating cellular elements which at that stage cannot yet be attributed to a defined retinal cell type. In the adult stage the surface-located retinal immunoreactivity is assigned to Müller cells with strongest fluorescence at the apical and microvilli containing cell compartment. Also the cellular processes in the outer nuclear layer, the outer plexiform layer and to a minor extent the inner nuclear layer revealed immunoreactivity. 5'-Nucleotidase immunoreactivity was found at the ventricular surface also of the adult brain. There it was associated with the apical surface of ependymal cells. Our results suggest that 5'-nucleotidase is of general functional importance for the metabolism of nucleotides at the ventricular surface of the retina as well as the ventricles of the brain, a feature that is maintained throughout development. Müller cells thus share not only functional characteristics of astrocytes and oligodendrocytes, as previously revealed, but also of ependymal cells to which they are closely related.
J Neurochem, 2002
Synaptic vesicles isolated from electric ray electric organ have been shown previously to contain... more Synaptic vesicles isolated from electric ray electric organ have been shown previously to contain a 34-kDa protein that binds azido-ATP, azido-AMP, and N-ethylmaleimide. The protein was found to share similarities with the mitochondrial ADP/ATP carrier and assumed to represent the synaptic vesicle nucleotide transporter. Synaptic vesicles were purified by sucrose density gradient centrifugation and subsequent chromatography on Sephacryl S-1000 from both Torpedo electric organ and bovine brain cerebral cortex. They contained ATP-binding proteins of 35 kDa and 34 kDa, respectively. ATP binding was inhibited by AMP. Both proteins were highly enriched after column chromatography of vesicle proteins on AMP-Sepharose. Antibodies were obtained against both proteins. Antibodies against the bovine brain synaptic vesicle protein of 34 kDa bound specifically to the 35-kDa protein of Torpedo vesicles. An N-terminal sequence obtained against the 34-kDa protein of bovine brain synaptic vesicles identified it as glyceraldehyde-3-phosphate dehydrogenase. The previously observed molecular characteristics of the putative vesicular nucleotide transporter in Torpedo fit those of glyceraldehyde-3-phosphate dehydrogenase. We, therefore, suggest that the protein previously identified as putative nucleotide transporter is, in fact, glyceraldehyde-3-phosphate dehydrogenase.
J Neurochem, 2001
Ecto-59-nucleotidase is regarded as being the key enzyme in the formation of the neuromodulator a... more Ecto-59-nucleotidase is regarded as being the key enzyme in the formation of the neuromodulator aden- osine from released ATP. However, the association of ecto-59-nucleotidase with nerve terminals is not consen- sual. Only enzyme histochemical and biochemical stud- ies, but not immunocytochemical studies, agree on a general synaptic location of the enzyme. To clarify this issue further we tested the effect of an antibody against ecto-59-nucleotidase, previously used in immunocyto- chemical studies, on the activity of ecto-59-nucleotidase in fractions of nerve terminals isolated from different ar- eas of rat hippocampus. The specific activity of extracel- lular AMP catabolism was higher in synaptosomes from the CA3 area (0.81 6 0.06 nmol/min/mg of protein) than from synaptosomes from the CA1 area or the dentate gyrus or from the whole hippocampus (0.49 - 0.68 nmol/ min/mg of protein). The catabolism of AMP (10 mM) was equally inhibited (85-92%) in synaptosomes from whole hippocampus, CA1, CA3, or dentate gyrus by a,b-meth- ylene-ADP (100 mM) and equally unaffected by p-nitro- phenyl phosphate (0.5 mM) or rabbit IgGs (100 mg/ml). However, the antiserum against ecto-59-nucleotidase (100 mg/ml) inhibited extracellular AMP catabolism by 44% in CA3 synaptosomes but had little or no effect in synaptosomes from CA1, dentate gyrus, or whole hip- pocampus. A similar difference in the inhibitory potential of the antibody was observed between fractions of iso- lated 59-nucleotidase binding to concanavalin A-Sepha- rose (70%) and fractions not retained by the lectin col- umn (18%). Taken together, these results suggest that immunological isoforms of ecto-59-nucleotidase exist in the rat hippocampal nerve terminals, with predominance in the CA3 area. Key Words: Nerve terminals—Ecto-59- nucleotidase—Adenosine—Ecto-nucleotidases. J. Neurochem. 74, 334 -338 (2000). Adenosine is an important neuromodulator in the ner- vous system. Inhibition or facilitation of synaptic trans- mission via A1 and A2A receptors, respectively, depends on the rate of extracellular adenosine formation (Correia- de-Saet al., 1996; Cunha et al., 1996 a). Adenosine can be released directly through bidirectional nonconcentra- tive adenosine transporters, or it can originate from re- leased adenine nucleotides by extracellular catabolism
Ecto-nucleotidases, molecular properties and functional impact Recibido el 15 de marzo de 2007
Cell and Tissue Research, Mar 31, 1995
5'-Nucleotidase hydrolyzes 5"-mononucleotides to their nucleosides but is also thought to have a ... more 5'-Nucleotidase hydrolyzes 5"-mononucleotides to their nucleosides but is also thought to have a function in neuronal differentiation and synapse formation. The distribution of the enzyme, a glycosyl-phosphatidylinositol-anchored sialoglycoprotein, was investigated in PC12 cells using immunofluorescence microscopy. 5'-Nucleotidase was located both in intracellular compartments and at the cell surface. There was no principal difference in the cellular distribution between undifferentiated cells and after neuritogenic differentiation by nerve growth factor. Intracellularly, 5'-nucleotidase often revealed a sickle-shaped perinuclear distribution and a dotted pattern throughout the cytoplasm, including that of neurites and growth cones. The intracellular distribution was clearly different from that of the synaptic vesicle protein synaptophysin. However, the dotted fluorescence resembled that obtained after uptake of the endosomal marker acridine orange. 5'-Nucleotidase was present on the entire cell surface including all neurites formed after differentiation. There was no increase in 5'nucleotidase fluorescence at synapse-like contacts between the tips of neurites and other PC12 cells. Surfacelocated 5'-nucleotidase could no longer be detected after the application of glycosyl-phosphatidylinositol-specific phospholipase C to cultured cells. This treatment did not affect PC12 cell differentiation. Our results thus reveal 5'-nucleotidase both at the surface and within organelles and suggest that PC12 cells may be used as a model system for the study of the physiological function of 5'-nucleotidase in neural cells.
Journal of Medicinal Chemistry, 2009
Prodrugs of adenosine A(2A) receptor agonists were developed that are activated by ecto-5&... more Prodrugs of adenosine A(2A) receptor agonists were developed that are activated by ecto-5'-nucleotidase (ecto-5'-NT, CD73). Because ecto-5'-NT is upregulated in inflamed tissue, the A(2A) agonists are expected to be released from their prodrug form at the sites of inflammation. 2-(Ar)alkyl-substituted AMP derivatives were synthesized and investigated. Certain 2-substituted AMP derivatives, including 2-hexylthio-AMP, 2-cyclopentylthio-AMP, 2-cyclohexylmethylthio-AMP, and 2-cyclohexylethylthio-AMP were accepted as substrates by ecto-5'-NT and readily converted to the corresponding 2-substituted adenosine derivatives. The 2-cyclohexylethylthio substitution was a good compromise between the requirements of the ecto-5'-NT and the adenosine A(2A) receptor. The corresponding AMP derivative (12g) was a similarly good substrate as AMP itself, while the resulting adenosine derivative (11g) was a relatively potent A(2A) agonist (radioligand binding to rat brain striatal membranes: K(i) = 372 nM; inhibition of anti-CD3/anti-CD28-induced IFN-gamma release in mouse CD4+ cells: EC(50) = 50 nM). Compound 11g was released from 12g by incubation with CD4+ cells isolated from wild-type mice but only to a much smaller extent by cells from ecto-5'-NT knockout mice. Compound 12g will be a new lead structure for the development of more potent and selective ecto-5'-NT-activated prodrugs of selective anti-inflammatory A(2A) receptor agonists.
Anales De La Real Academia Nacional De Farmacia, 2007
Pflugers Archiv European Journal of Physiology, Apr 25, 2006
The development of the nervous system requires complex series of cellular programming and interce... more The development of the nervous system requires complex series of cellular programming and intercellular communication events that lead from the early neural induction to the formation of a highly structured central and peripheral nervous system. Neurogenesis continuously takes place also in select regions of the adult mammalian brain. During the past years, a multiplicity of cellular control mechanisms has been identified, ranging from differential transcriptional mediators to inducers or inhibitors of cell specification or neurite outgrowth. While the identification of transcription factors typical for the stage-specific progression has been a topic of key interest for many years, less is known concerning the potential multiplicity of relevant intercellular signaling pathways and the fine tuning of epigenetic gene regulation. Nucleotide receptors can induce a multiplicity of cellular signaling pathways and are involved in multiple molecular interactions, thus opening the possibility of cross talk between several signaling pathways, including growth factors, cytokines, and extracellular matrix components. An increasing number of studies provides evidence for a role of nucleotide signaling in nervous system development. This includes progenitor cell proliferation, cell migration, neuronal and glial cellular interaction and differentiation, and synaptic network formation.
J Med Chem, 2009
Prodrugs of adenosine A(2A) receptor agonists were developed that are activated by ecto-5&... more Prodrugs of adenosine A(2A) receptor agonists were developed that are activated by ecto-5'-nucleotidase (ecto-5'-NT, CD73). Because ecto-5'-NT is upregulated in inflamed tissue, the A(2A) agonists are expected to be released from their prodrug form at the sites of inflammation. 2-(Ar)alkyl-substituted AMP derivatives were synthesized and investigated. Certain 2-substituted AMP derivatives, including 2-hexylthio-AMP, 2-cyclopentylthio-AMP, 2-cyclohexylmethylthio-AMP, and 2-cyclohexylethylthio-AMP were accepted as substrates by ecto-5'-NT and readily converted to the corresponding 2-substituted adenosine derivatives. The 2-cyclohexylethylthio substitution was a good compromise between the requirements of the ecto-5'-NT and the adenosine A(2A) receptor. The corresponding AMP derivative (12g) was a similarly good substrate as AMP itself, while the resulting adenosine derivative (11g) was a relatively potent A(2A) agonist (radioligand binding to rat brain striatal membranes: K(i) = 372 nM; inhibition of anti-CD3/anti-CD28-induced IFN-gamma release in mouse CD4+ cells: EC(50) = 50 nM). Compound 11g was released from 12g by incubation with CD4+ cells isolated from wild-type mice but only to a much smaller extent by cells from ecto-5'-NT knockout mice. Compound 12g will be a new lead structure for the development of more potent and selective ecto-5'-NT-activated prodrugs of selective anti-inflammatory A(2A) receptor agonists.
Cell Tissue Res, 1989
Binding sites for antibodies against membrane proteins of synaptic vesicles have been shown to be... more Binding sites for antibodies against membrane proteins of synaptic vesicles have been shown to be enhanced at nodes of Ranvier in electromotor axons of the electric ray Torpedo marmorata and sciatic nerve axons of the rat, using indirect immunofluorescence and monoclonal antibodies against the synaptic vesicle transmembrane proteins SV2 and synaptophysin (rat) or SV2 (Torpedo). In the electric lobe of Torpedo, vesicle-membrane constituents occurred at higher density in the proximal axon segments covered by oligodendroglia cells than in the distal axon segments where myelin is formed by Schwann cells. Antibody binding sites were enhanced at nodes forming the borderline of the central and peripheral nervous systems. Filamentous actin was present in the Schwann-cell processes covering both the nodal and the paranodal axon segments as suggested by the pattern of phalloidin labelling. Furthermore, in rat sciatic nerve, Schmidt-Lanterman incisures were intensely labelled by phalloidin. A similar nodal distribution was found for binding sites of antibodies against actin and myosin. Binding of antibodies to tubulin was enhanced at nodes in Torpedo electromotor axons. The apparent nodal accumulation of constituents of synaptic vesicle membranes and the presence of filamentous actin and of myosin are discussed in relation to the substantial constriction of the axoplasm at nodes of Ranvier.
Cell and tissue research, Jul 26, 2016
Ecto-5'-nucleotidase (eN) is the major extracellular adenosine-producing ecto-enzyme in mouse... more Ecto-5'-nucleotidase (eN) is the major extracellular adenosine-producing ecto-enzyme in mouse brain. Via the production of adenosine, eN participates in many physiological and pathological processes, such as wakefulness, inflammation, nociception and neuroprotection. The mechanisms regulating the expression of eN are therefore of considerable neurobiological and clinical interest. Having previously described a modulatory effect of melatonin in the regulation of eN mRNA levels, we decided to analyze the melatonin receptor subtype involved in the regulation of eN mRNA levels by comparing eN mRNA patterns in melatonin-proficient transgenic mice lacking either the melatonin receptor subtype 1 (MT1 KO) or both melatonin receptor subtypes (MT1 and MT2; MT1/2 KO) with the corresponding melatonin-proficient wild-type (WT) controls. By means of radioactive in situ hybridization, eN mRNA levels were found to be diminished in both MT1 and MT1/2 KO mice compared with WT controls suggesting ...
Cellular and Molecular Neurobiology, May 1, 2002
1. Cultured astrocytes cells release a variety of low and high molecular weight messenger substan... more 1. Cultured astrocytes cells release a variety of low and high molecular weight messenger substances and express proteins of the exocytotic pathway including synaptic SNARE proteins. For analyzing the molecular mechanisms of astrocytic messenger release, permanent cell lines with astrocytic properties would provide useful tools. 2. We analyzed the potential of the human malignant astrocytoma-derived cell line U373 MG to express proteins involved in regulated exo-and endocytosis. An immunoblot analysis identified the astrocyte marker glial fibrillary acidic protein, microtubule-associated protein 2, the v-SNAREs VAMP I, VAMP II, and cellubrevin and the t-SNAREs syntaxin I, SNAP-23, and SNAP-25. 3. The cells also express the secretory granule protein secretogranin II. Although secretogranin II immunofluorescence reveals larger fluorescence spots, the majority of the SNARE proteins is associated with smaller organelles. The immunofluorescence is distributed throughout the cytoplasm and accumulates at processes and the growing edges of cells. 4. The organellar association of SNARE proteins was confirmed by heterologous expression of recombinant fusion proteins. Following subcellular fractionation organelles of lower buoyant density carried the majority of VAMP II. Secretogranin II was associated with organelles of high buoyant density containing a small contribution of VAMP II. 5. The results suggest that U373 MG cells have in common a considerable number of properties with long-term cultured astrocytes rather than with cultured oligodendrocytes or neurons. They contain two types of organelles that can be physically separated and may be employed in the differential release of messengers.
Progress in Brain Research, 1996
Purinergic Signalling
Geoffrey Burnstock will be remembered as the scientist who set up an entirely new field of interc... more Geoffrey Burnstock will be remembered as the scientist who set up an entirely new field of intercellular communication, signaling via nucleotides. The signaling cascades involved in purinergic signaling include intracellular storage of nucleotides, nucleotide release, extracellular hydrolysis, and the effect of the released compounds or their hydrolysis products on target tissues via specific receptor systems. In this context ectonucleotidases play several roles. They inactivate released and physiologically active nucleotides, produce physiologically active hydrolysis products, and facilitate nucleoside recycling. This review briefly highlights the development of our knowledge of two types of enzymes involved in extracellular nucleotide hydrolysis and thus purinergic signaling, the ectonucleoside triphosphate diphosphohydrolases, and ecto-5′-nucleotidase.
Purinergic Signalling
Professor Geoffrey Burnstock, a great luminary in science and the founder of our field of puriner... more Professor Geoffrey Burnstock, a great luminary in science and the founder of our field of purinergic signalling, sadly but peacefully died in Melbourne, Australia, on the 2nd of June, 2020, at the age of 91. He had retired at the age of 88, in October 2017, when, after 42 years in London, he decided to fulfill his wife's and family's desire to move back to Australia, where, in 1959, he had been appointed Senior Lecturer at Melbourne University, his first important academic engagement. Geoffrey Burnstock grew up in London where he was born on May 10, 1929. After completing his secondary education at Greenford County Grammar School in 1946, he did his National service with the Air Force in 1947. In 1953, he completed a BSc degree at King's College, University of London, majoring in mathematics and physics. He subsequently completed a PhD at King's College and University College London in the field of zoology, studying gut motility in fish (1957). This turned out to be a great start for the young scientist as his first paper ever was published in Nature. In 1956, he moved to the Physiology Department at the National Institute for Medical Research in Mill Hill, London, as a postdoctoral fellow with Wilhelm Feldberg (1956-1957). There, he developed a novel technique for recording membrane activities from smooth muscle, the "sucrose gap technique." This led to a position with Edith Bulbring in the
Frontiers in Cellular Neuroscience
In mammalian species, including humans, the hippocampal dentate gyrus (DG) is a primary region of... more In mammalian species, including humans, the hippocampal dentate gyrus (DG) is a primary region of adult neurogenesis. Aberrant adult hippocampal neurogenesis is associated with neurological pathologies. Understanding the cellular mechanisms controlling adult hippocampal neurogenesis is expected to open new therapeutic strategies for mental disorders. Microglia is intimately associated with neural progenitor cells in the hippocampal DG and has been implicated, under varying experimental conditions, in the control of the proliferation, differentiation and survival of neural precursor cells. But the underlying mechanisms remain poorly defined. Using fluorescent in situ hybridization we show that microglia in brain express the ADP-activated P2Y 13 receptor under basal conditions and that P2ry13 mRNA is absent from neurons, astrocytes, and neural progenitor cells. Disrupting P2ry13 decreases structural complexity of microglia in the hippocampal subgranular zone (SGZ). But it increases progenitor cell proliferation and new neuron formation. Our data suggest that P2Y 13 receptor-activated microglia constitutively attenuate hippocampal neurogenesis. This identifies a signaling pathway whereby microglia, via a nucleotide-mediated mechanism, contribute to the homeostatic control of adult hippocampal neurogenesis. Selective P2Y 13 R antagonists could boost neurogenesis in pathological conditions associated with impaired hippocampal neurogenesis.
e-Neuroforum
ZusammenfassungATP ist eines der vielseitigsten zellulären Moleküle überhaupt. Seine Rolle als ex... more ZusammenfassungATP ist eines der vielseitigsten zellulären Moleküle überhaupt. Seine Rolle als extrazellulärer Signalstoff wurde erst in den 90er Jahren des letzten Jahrhunderts konsolidiert, mit der Klonierung der Rezeptoren (P2-Rezeptoren) und von Enzymen (Ekto- Nukleotidasen), die extrazelluläres ATP hydrolysieren können. P2-Rezeptoren können je nach Subtyp durch ATP oder durch ADP, UTP, UDP und verschiedene Diadenosinpolyphosphate aktiviert werden. ATP und andere Nukleotide werden in Subtypen synaptischer Vesikel gespeichert und können exozytotisch freigesetzt werden. Außerhalb der Zelle werden Nukleotide bis zum jeweiligen Nukleosid hydrolysiert. Extrazellulär gebildetes Adenosin aktiviert seinerseits P1-Rezeptoren und wird anschließend wieder in die Nervenendigung oder in benachbarte Zellen aufgenommen. Bis jetzt wurden mehrere Ekto-Nukleotidasen kloniert und funktionell charakterisiert. Gegenwärtig analysieren wir ihre unterschiedliche zelluläre Lokalisierung im zentralen und...
Developmental Brain Research, Aug 28, 1995
The distribution of 5'-nucleotidase was investigated in the developing mouse retina and a... more The distribution of 5'-nucleotidase was investigated in the developing mouse retina and also in the retina of the adult mouse and rat using a monospecific antibody directed against 5'-nucleotidase isolated from bovine brain. Already at the stage of the formation of the eye cup strong immunofluorescence was found at the ventricular surface. Throughout development the enzyme remained associated with the outer surface of the retina that corresponds to the former inner surface of the eye vesicle. During embryonic development immunoreactivity was also associated with the proliferating cellular elements which at that stage cannot yet be attributed to a defined retinal cell type. In the adult stage the surface-located retinal immunoreactivity is assigned to Müller cells with strongest fluorescence at the apical and microvilli containing cell compartment. Also the cellular processes in the outer nuclear layer, the outer plexiform layer and to a minor extent the inner nuclear layer revealed immunoreactivity. 5'-Nucleotidase immunoreactivity was found at the ventricular surface also of the adult brain. There it was associated with the apical surface of ependymal cells. Our results suggest that 5'-nucleotidase is of general functional importance for the metabolism of nucleotides at the ventricular surface of the retina as well as the ventricles of the brain, a feature that is maintained throughout development. Müller cells thus share not only functional characteristics of astrocytes and oligodendrocytes, as previously revealed, but also of ependymal cells to which they are closely related.
J Neurochem, 2002
Synaptic vesicles isolated from electric ray electric organ have been shown previously to contain... more Synaptic vesicles isolated from electric ray electric organ have been shown previously to contain a 34-kDa protein that binds azido-ATP, azido-AMP, and N-ethylmaleimide. The protein was found to share similarities with the mitochondrial ADP/ATP carrier and assumed to represent the synaptic vesicle nucleotide transporter. Synaptic vesicles were purified by sucrose density gradient centrifugation and subsequent chromatography on Sephacryl S-1000 from both Torpedo electric organ and bovine brain cerebral cortex. They contained ATP-binding proteins of 35 kDa and 34 kDa, respectively. ATP binding was inhibited by AMP. Both proteins were highly enriched after column chromatography of vesicle proteins on AMP-Sepharose. Antibodies were obtained against both proteins. Antibodies against the bovine brain synaptic vesicle protein of 34 kDa bound specifically to the 35-kDa protein of Torpedo vesicles. An N-terminal sequence obtained against the 34-kDa protein of bovine brain synaptic vesicles identified it as glyceraldehyde-3-phosphate dehydrogenase. The previously observed molecular characteristics of the putative vesicular nucleotide transporter in Torpedo fit those of glyceraldehyde-3-phosphate dehydrogenase. We, therefore, suggest that the protein previously identified as putative nucleotide transporter is, in fact, glyceraldehyde-3-phosphate dehydrogenase.
J Neurochem, 2001
Ecto-59-nucleotidase is regarded as being the key enzyme in the formation of the neuromodulator a... more Ecto-59-nucleotidase is regarded as being the key enzyme in the formation of the neuromodulator aden- osine from released ATP. However, the association of ecto-59-nucleotidase with nerve terminals is not consen- sual. Only enzyme histochemical and biochemical stud- ies, but not immunocytochemical studies, agree on a general synaptic location of the enzyme. To clarify this issue further we tested the effect of an antibody against ecto-59-nucleotidase, previously used in immunocyto- chemical studies, on the activity of ecto-59-nucleotidase in fractions of nerve terminals isolated from different ar- eas of rat hippocampus. The specific activity of extracel- lular AMP catabolism was higher in synaptosomes from the CA3 area (0.81 6 0.06 nmol/min/mg of protein) than from synaptosomes from the CA1 area or the dentate gyrus or from the whole hippocampus (0.49 - 0.68 nmol/ min/mg of protein). The catabolism of AMP (10 mM) was equally inhibited (85-92%) in synaptosomes from whole hippocampus, CA1, CA3, or dentate gyrus by a,b-meth- ylene-ADP (100 mM) and equally unaffected by p-nitro- phenyl phosphate (0.5 mM) or rabbit IgGs (100 mg/ml). However, the antiserum against ecto-59-nucleotidase (100 mg/ml) inhibited extracellular AMP catabolism by 44% in CA3 synaptosomes but had little or no effect in synaptosomes from CA1, dentate gyrus, or whole hip- pocampus. A similar difference in the inhibitory potential of the antibody was observed between fractions of iso- lated 59-nucleotidase binding to concanavalin A-Sepha- rose (70%) and fractions not retained by the lectin col- umn (18%). Taken together, these results suggest that immunological isoforms of ecto-59-nucleotidase exist in the rat hippocampal nerve terminals, with predominance in the CA3 area. Key Words: Nerve terminals—Ecto-59- nucleotidase—Adenosine—Ecto-nucleotidases. J. Neurochem. 74, 334 -338 (2000). Adenosine is an important neuromodulator in the ner- vous system. Inhibition or facilitation of synaptic trans- mission via A1 and A2A receptors, respectively, depends on the rate of extracellular adenosine formation (Correia- de-Saet al., 1996; Cunha et al., 1996 a). Adenosine can be released directly through bidirectional nonconcentra- tive adenosine transporters, or it can originate from re- leased adenine nucleotides by extracellular catabolism
Ecto-nucleotidases, molecular properties and functional impact Recibido el 15 de marzo de 2007
Cell and Tissue Research, Mar 31, 1995
5'-Nucleotidase hydrolyzes 5"-mononucleotides to their nucleosides but is also thought to have a ... more 5'-Nucleotidase hydrolyzes 5"-mononucleotides to their nucleosides but is also thought to have a function in neuronal differentiation and synapse formation. The distribution of the enzyme, a glycosyl-phosphatidylinositol-anchored sialoglycoprotein, was investigated in PC12 cells using immunofluorescence microscopy. 5'-Nucleotidase was located both in intracellular compartments and at the cell surface. There was no principal difference in the cellular distribution between undifferentiated cells and after neuritogenic differentiation by nerve growth factor. Intracellularly, 5'-nucleotidase often revealed a sickle-shaped perinuclear distribution and a dotted pattern throughout the cytoplasm, including that of neurites and growth cones. The intracellular distribution was clearly different from that of the synaptic vesicle protein synaptophysin. However, the dotted fluorescence resembled that obtained after uptake of the endosomal marker acridine orange. 5'-Nucleotidase was present on the entire cell surface including all neurites formed after differentiation. There was no increase in 5'nucleotidase fluorescence at synapse-like contacts between the tips of neurites and other PC12 cells. Surfacelocated 5'-nucleotidase could no longer be detected after the application of glycosyl-phosphatidylinositol-specific phospholipase C to cultured cells. This treatment did not affect PC12 cell differentiation. Our results thus reveal 5'-nucleotidase both at the surface and within organelles and suggest that PC12 cells may be used as a model system for the study of the physiological function of 5'-nucleotidase in neural cells.
Journal of Medicinal Chemistry, 2009
Prodrugs of adenosine A(2A) receptor agonists were developed that are activated by ecto-5&... more Prodrugs of adenosine A(2A) receptor agonists were developed that are activated by ecto-5'-nucleotidase (ecto-5'-NT, CD73). Because ecto-5'-NT is upregulated in inflamed tissue, the A(2A) agonists are expected to be released from their prodrug form at the sites of inflammation. 2-(Ar)alkyl-substituted AMP derivatives were synthesized and investigated. Certain 2-substituted AMP derivatives, including 2-hexylthio-AMP, 2-cyclopentylthio-AMP, 2-cyclohexylmethylthio-AMP, and 2-cyclohexylethylthio-AMP were accepted as substrates by ecto-5'-NT and readily converted to the corresponding 2-substituted adenosine derivatives. The 2-cyclohexylethylthio substitution was a good compromise between the requirements of the ecto-5'-NT and the adenosine A(2A) receptor. The corresponding AMP derivative (12g) was a similarly good substrate as AMP itself, while the resulting adenosine derivative (11g) was a relatively potent A(2A) agonist (radioligand binding to rat brain striatal membranes: K(i) = 372 nM; inhibition of anti-CD3/anti-CD28-induced IFN-gamma release in mouse CD4+ cells: EC(50) = 50 nM). Compound 11g was released from 12g by incubation with CD4+ cells isolated from wild-type mice but only to a much smaller extent by cells from ecto-5'-NT knockout mice. Compound 12g will be a new lead structure for the development of more potent and selective ecto-5'-NT-activated prodrugs of selective anti-inflammatory A(2A) receptor agonists.
Anales De La Real Academia Nacional De Farmacia, 2007
Pflugers Archiv European Journal of Physiology, Apr 25, 2006
The development of the nervous system requires complex series of cellular programming and interce... more The development of the nervous system requires complex series of cellular programming and intercellular communication events that lead from the early neural induction to the formation of a highly structured central and peripheral nervous system. Neurogenesis continuously takes place also in select regions of the adult mammalian brain. During the past years, a multiplicity of cellular control mechanisms has been identified, ranging from differential transcriptional mediators to inducers or inhibitors of cell specification or neurite outgrowth. While the identification of transcription factors typical for the stage-specific progression has been a topic of key interest for many years, less is known concerning the potential multiplicity of relevant intercellular signaling pathways and the fine tuning of epigenetic gene regulation. Nucleotide receptors can induce a multiplicity of cellular signaling pathways and are involved in multiple molecular interactions, thus opening the possibility of cross talk between several signaling pathways, including growth factors, cytokines, and extracellular matrix components. An increasing number of studies provides evidence for a role of nucleotide signaling in nervous system development. This includes progenitor cell proliferation, cell migration, neuronal and glial cellular interaction and differentiation, and synaptic network formation.
J Med Chem, 2009
Prodrugs of adenosine A(2A) receptor agonists were developed that are activated by ecto-5&... more Prodrugs of adenosine A(2A) receptor agonists were developed that are activated by ecto-5'-nucleotidase (ecto-5'-NT, CD73). Because ecto-5'-NT is upregulated in inflamed tissue, the A(2A) agonists are expected to be released from their prodrug form at the sites of inflammation. 2-(Ar)alkyl-substituted AMP derivatives were synthesized and investigated. Certain 2-substituted AMP derivatives, including 2-hexylthio-AMP, 2-cyclopentylthio-AMP, 2-cyclohexylmethylthio-AMP, and 2-cyclohexylethylthio-AMP were accepted as substrates by ecto-5'-NT and readily converted to the corresponding 2-substituted adenosine derivatives. The 2-cyclohexylethylthio substitution was a good compromise between the requirements of the ecto-5'-NT and the adenosine A(2A) receptor. The corresponding AMP derivative (12g) was a similarly good substrate as AMP itself, while the resulting adenosine derivative (11g) was a relatively potent A(2A) agonist (radioligand binding to rat brain striatal membranes: K(i) = 372 nM; inhibition of anti-CD3/anti-CD28-induced IFN-gamma release in mouse CD4+ cells: EC(50) = 50 nM). Compound 11g was released from 12g by incubation with CD4+ cells isolated from wild-type mice but only to a much smaller extent by cells from ecto-5'-NT knockout mice. Compound 12g will be a new lead structure for the development of more potent and selective ecto-5'-NT-activated prodrugs of selective anti-inflammatory A(2A) receptor agonists.
Cell Tissue Res, 1989
Binding sites for antibodies against membrane proteins of synaptic vesicles have been shown to be... more Binding sites for antibodies against membrane proteins of synaptic vesicles have been shown to be enhanced at nodes of Ranvier in electromotor axons of the electric ray Torpedo marmorata and sciatic nerve axons of the rat, using indirect immunofluorescence and monoclonal antibodies against the synaptic vesicle transmembrane proteins SV2 and synaptophysin (rat) or SV2 (Torpedo). In the electric lobe of Torpedo, vesicle-membrane constituents occurred at higher density in the proximal axon segments covered by oligodendroglia cells than in the distal axon segments where myelin is formed by Schwann cells. Antibody binding sites were enhanced at nodes forming the borderline of the central and peripheral nervous systems. Filamentous actin was present in the Schwann-cell processes covering both the nodal and the paranodal axon segments as suggested by the pattern of phalloidin labelling. Furthermore, in rat sciatic nerve, Schmidt-Lanterman incisures were intensely labelled by phalloidin. A similar nodal distribution was found for binding sites of antibodies against actin and myosin. Binding of antibodies to tubulin was enhanced at nodes in Torpedo electromotor axons. The apparent nodal accumulation of constituents of synaptic vesicle membranes and the presence of filamentous actin and of myosin are discussed in relation to the substantial constriction of the axoplasm at nodes of Ranvier.
Cell and tissue research, Jul 26, 2016
Ecto-5'-nucleotidase (eN) is the major extracellular adenosine-producing ecto-enzyme in mouse... more Ecto-5'-nucleotidase (eN) is the major extracellular adenosine-producing ecto-enzyme in mouse brain. Via the production of adenosine, eN participates in many physiological and pathological processes, such as wakefulness, inflammation, nociception and neuroprotection. The mechanisms regulating the expression of eN are therefore of considerable neurobiological and clinical interest. Having previously described a modulatory effect of melatonin in the regulation of eN mRNA levels, we decided to analyze the melatonin receptor subtype involved in the regulation of eN mRNA levels by comparing eN mRNA patterns in melatonin-proficient transgenic mice lacking either the melatonin receptor subtype 1 (MT1 KO) or both melatonin receptor subtypes (MT1 and MT2; MT1/2 KO) with the corresponding melatonin-proficient wild-type (WT) controls. By means of radioactive in situ hybridization, eN mRNA levels were found to be diminished in both MT1 and MT1/2 KO mice compared with WT controls suggesting ...
Cellular and Molecular Neurobiology, May 1, 2002
1. Cultured astrocytes cells release a variety of low and high molecular weight messenger substan... more 1. Cultured astrocytes cells release a variety of low and high molecular weight messenger substances and express proteins of the exocytotic pathway including synaptic SNARE proteins. For analyzing the molecular mechanisms of astrocytic messenger release, permanent cell lines with astrocytic properties would provide useful tools. 2. We analyzed the potential of the human malignant astrocytoma-derived cell line U373 MG to express proteins involved in regulated exo-and endocytosis. An immunoblot analysis identified the astrocyte marker glial fibrillary acidic protein, microtubule-associated protein 2, the v-SNAREs VAMP I, VAMP II, and cellubrevin and the t-SNAREs syntaxin I, SNAP-23, and SNAP-25. 3. The cells also express the secretory granule protein secretogranin II. Although secretogranin II immunofluorescence reveals larger fluorescence spots, the majority of the SNARE proteins is associated with smaller organelles. The immunofluorescence is distributed throughout the cytoplasm and accumulates at processes and the growing edges of cells. 4. The organellar association of SNARE proteins was confirmed by heterologous expression of recombinant fusion proteins. Following subcellular fractionation organelles of lower buoyant density carried the majority of VAMP II. Secretogranin II was associated with organelles of high buoyant density containing a small contribution of VAMP II. 5. The results suggest that U373 MG cells have in common a considerable number of properties with long-term cultured astrocytes rather than with cultured oligodendrocytes or neurons. They contain two types of organelles that can be physically separated and may be employed in the differential release of messengers.
Progress in Brain Research, 1996